Drosophila has been successfully used to study regulatory genetic networks of inductive signals and transcription factors that determine cardiac specification and morphogenesis, but the genetic control of cardiac physiology is only beginning to be investigated. Using a cardiac performance-based genetic screen, we discovered a novel genetic interaction between Cdc42 and the NK homeobox gene tinman in regulating cardiac function in the fly. We have identified a heretofore-unknown requirement for the Rho GTPase Cdc42 in regulating the establishment of cardiac function in the adult fly. Significantly, this interaction is conserved in mice, where combined reduction of Cdc42 and Nkx2-5 caused defects in heart contraction, rhythm, and electrophysiological function. Based on the discovery of the interaction between this Rho GTPase with a cardiogenic transcription factor in maintaining proper cardiac function, it is conceivable that other genetic interactors and potential polygenic contributors to heart disease traits can be identified using the fly as a screening platform.
Cdc42 is a well-characterized GTPase, and has been widely studied in different organs and tissues, ranging from worms to humans. Because Cdc42
was shown to be involved in regulation of the actin/myosin cytoskeleton in the cells of various organisms, we reasoned that Cdc42
might also control cardiac cell morphogenesis. In Drosophila
and mouse we found that complete loss of Cdc42
in the heart leads to defects in cardiac morphogenesis (unpublished data). In the adult heart, Cdc42 was previously shown to be associated with cardiac myocyte hypertrophy (Clerk and Sugden, 2000
; Pandur et al., 2002
; Sellin et al., 2006
). Recent work shows that Cdc42 plays a protective role against hypertrophy, as conditional deletion of Cdc42 in the adult heart led to greater cardiac hypertrophy and increased incidence of heart failure (Maillet et al., 2009
). Interestingly, our findings show that interference with Cdc42
function in the adult fly heart via expression of a dominant-negative form of Cdc42
is sufficient to induce functional and morphological defects, suggesting that, in addition to interacting with tinman
itself is essential for maintaining normal adult heart structure and function. The specific differences between the fly and the mouse phenotypes in these studies may be attributed to differences in assays as well as to evolutionary changes of Cdc42
function from insects to vertebrates. However, the fact that Cdc42
adult-specific mutant fly hearts are defective in Z-line/myofibril assembly is consistent with a previous report in a tissue culture model for cardiac hypertrophy showing that sarcomere units are misassembled in cultured rat cardiomyocytes when Cdc42
expression is disrupted (Nagai et al., 2003
; Brown et al., 2006
). In addition, Cdc42
interacts with a known downstream effector of Cdc42
, which participates in actin filament assembly and dynamics (Harden et al., 1996
; Bahri et al., 2010
), thus consistent with a possible role for Cdc42
in myofibrillar organization.
Similar to our Cdc42;Nkx2-5
compound heterozygote analysis, it has been shown that mice transheterozygous for loss-of-function alleles of Nkx2-5
also exhibit a synergistic interaction, in particular with respect to the development of the cardiac conduction system (Moskowitz et al., 2007
). It is therefore likely that other cardiac transcription factors in combination with new genes will reveal similar patterns of interactions in flies and mammals. Although different model organisms have been used to study cardiac disease, the ability to investigate polygenic traits underlying these diseases in a systematic fashion in the adult is limited. Here, we provide evidence that rigorous screening for modifiers of heart disease can be achieved using Drosophila
, leading to identification of similar polygenic trait interactions in mammals.
The genetic relationship between fly Cdc42
with the cardiac determinant tinman
in regulating adult heart contraction may occur, in part, via modulation of potassium channel gene expression, dSUR
, which have previously been shown to be critical regulators of heart function (Johnson et al., 1998
; Akasaka et al., 2006
). Indeed, Cdc42-dSUR or –slo
transheterozygotes show a strong interaction reflected in increased arrhythmias. Interestingly, these transheterozygotes also exhibit disturbances in myofibrillar organization, suggesting these ion channel abnormalities somehow also produce sarcomeric defects, perhaps via a secondary structural remodeling effect. Although there is increasing evidence of such remodeling processes being affected by ion channel defects (unpublished data), the underlying mechanism is not yet understood.
A strong interaction was also observed between the mouse homologues of Cdc42 and Nkx2-5 in adult heart function. Most interestingly, double haploinsufficiency of Cdc42 and Nkx2-5 led to elongated QRS intervals, which is typical of abnormal conduction along the atrio-ventricular bundle, bundle branches, and Purkinje fibers. The observed elongated QT intervals in compound heterozygotes are also characteristic of long-QT syndrome in humans. These findings suggest a possible contribution of compound haploinsufficiency of Cdc42 and Nkx2-5 in human cardiac conduction diseases.
While attempting to elucidate the mechanistic link between Cdc42
, we found that miR-1 directly represses Cdc42
and that miR-1 itself can be repressed by Nkx2.5
expression. miR-1 is the first microRNA identified in the heart and plays a conserved role in cardiac morphogenesis, cell cycle, and conduction (Zhao et al., 2005
). miR-1 null mutant mice showed cardiac conduction defects including widened QRS complex and prolonged QT intervals (Zhao et al., 2007
), which is also affected in Cdc42;Nkx2-5
double-heterozygous mice. We speculate that loss or gain of miR-1 function causes an imbalance in gene expression in the heart, including that of Cdc42
. Decreasing the copy number of the direct miR-1 target Cdc42
, and that of a direct transcriptional miR-1 repressor, Nkx2-5, apparently destroys a delicate balance in the adult heart, tipping it toward deregulated cardiac function. This does not rule out that other mechanisms are also involved in the genetic interaction between Cdc42
. Because Cdc42
is a direct target of miR-1
and has a crucial function in modulating hypertrophic responses in the mouse, it will be interesting to test whether miR-1 and other microRNAs play a direct role in adult cardiac hypertrophy. Interestingly, Cdc42
was also found to be a target of miR-133, which is involved in cardiac hypertrophy (Carè et al., 2007
). Thus, the RhoGTPase Cdc42 appears to be directly regulated by two major cardiac microRNAs that are involved in controlling cardiac function and hypertrophy. The synergistic transcriptional activation of the dSUR
enhancer by Tinman and Cdc42 in vitro () is consistent with the idea that Cdc42 can act in a coactivating mechanism with Tinman, but this remains to be further investigated.
In conclusion, we have used the power of Drosophila genetics to uncover a new, conserved genetic pathway, linking genes with critical heart functions, tinman/Nkx2-5, miR-1, and Cdc42, to a novel network regulating adult heart physiology and performance. With the fly as a screening platform, it is thus possible to identify new polygenic contributors that ensure normal cardiac function relevant to human heart disease.